This application claims the priority of Korean Patent Application No. 2003-3669, filed on Jan. 20, 2003, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
1. Field of the Invention
The present invention relates to a microarray substrate having a patterned photoresist film with spot regions, a microarray, and a method of detecting a target material using the microarray.
2. Description of the Related Art
Generally, the term, “microarray” indicates an analysis system in which polymer molecules such as polynucleotides and proteins are immobilized in a high density on a solid substrate. The polymer molecules are immobilized on the spot regions which is arranged in an array. Such a microarray has been well known in the pertinent art. Examples of the microarray are disclosed in U.S. Pat. Nos. 5,445,934 and 5,744,305. Examples of the microarray include a protein array and a polynucleotide array. The term, “spot regions of the microarray” indicates regions on the microarray where polymer molecules such as proteins and polynucleotides are immobilized.
Generally, biological molecules, also called “probe molecules”, specifically bound to target materials, are mainly used as the polymer molecules immobilized on the microarray. The microarray has been usefully applied in a method of detecting target molecules and the like. According to an example of the method of detecting target molecules using a microarray, samples containing target nucleotides are spotted on the microarray having immobilized oligonucleotide probes thereon, followed by hybridization under a hybridization condition. After the hybridization is completed, the degree of hybridization is assessed by monitoring the intensity of a signal, such as light. Generally, the target molecules are labeled with detectable materials.
However, such a conventional microarray method for detecting target materials is accompanied by problems associated with nonspecific binding. As used herein, the term, “nonspecific binding” indicates the binding of target materials to other compounds on a microarray, for example, surface molecules of a substrate, in addition to probe molecules. Such nonspecific binding of target materials reduces the sensitivity of a signal to be measured, thereby causing a detection error.
As a conventional way to prevent the nonspecific binding of target materials, a method of coating a blocking material on a microarray during fabrication of the microarray or signal detection is used. For example, bovine serum albumin (BSA) is used as the blocking material. BSA serves to quench unreacted activated coupling compounds to minimize nonspecific binding. For this, BSA is coated in a monolayer on a solid substrate. However, the use of BSA may face with unexpected nonspecific binding problem according to the coating condition of BSA, for example, the concentration and duration of the coating. Also, immobilized molecules, i.e., probes may be distorted, which is known as “tailing”. In addition, there arises a problem in that compounds such as BSA are easily damaged by heat or chemicals.
FIG. 1 is a schematic diagram that illustrates a conventional microarray preparation method using BSA as a material for preventing the nonspecific binding of a target material. First, a coupling compound 10 is coated on a glass substrate 2. An example of the coupling compound is a silane compound with an end group such as aldehyde, epoxy, and amine. A primary antibody as a probe 12 is covalently bound to the coupling compound. Then, in order to prevent the nonspecific binding of the target material, BSA 14 is coated on the coupling compound as a blocking material for blocking the activation of the coupling compound unreacted with the probe 12. The microarray thus prepared can be used to directly detect an antigen which is the target material or to detect a secondary antibody using the antigen bound to the primary antibody.
As another method for preventing the nonspecific binding of a target material, in fabrication of a DNA chip, there is mainly used a method of coating an anionic chemical material on a solid substrate having an immobilized DNA probe thereon. However, this method also faces with a problem of lack of uniformity in terms that the degree of preventing the nonspecific binding varies depending on a coating condition. Also, the anionic chemical material is little used in a protein microarray because the activity of immobilized protein can be inhibited.
In addition, there is a method of using a probe containing polyethyleneglycol (PEG) or a polymer like PEG. However, this method has problems in that fabrication of the PEG-containing probe or a microarray containing the probe is complicated, a fabrication unit cost is high, and complete prevention of nonspecific binding is difficult.